Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

Regmi, Raju; Winkler, Pamina M.; Flauraud, Valentin; Borgman, Kyra J. E.; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme

doi:10.1021/acs.nanolett.7b02973

Cited by 51 publications

(64 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Two-component diffusion of TF-SM can be explained by its trapping behavior, which was observed previously with various methodologies and different SM analogues (38, 46–48). However, the two-component diffusion of cholesterol cannot be explained by simple trapping because one component is faster but not slower than the membrane diffusion of freely diffusing TF-PE.…”

Section: Resultssupporting

confidence: 53%

Nanoscale dynamics of cholesterol in the cell membrane

Pinkwart

Schneider

Lukoseviciute

et al. 2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

Cholesterol constitutes ∼30–40% of the mammalian plasma membrane, a larger fraction than of any other single component. It is a major player in numerous signaling processes as well as in shaping molecular membrane architecture. However, our knowledge of the dynamics of cholesterol in the plasma membrane is limited, restricting our understanding of the mechanisms regulating its involvement in cell signaling. Here, we applied advanced fluorescence imaging and spectroscopy approaches on in vitro (model membranes) and in vivo (live cells and embryos) membranes as well as in silico analysis to systematically study the nanoscale dynamics of cholesterol in biological membranes. Our results indicate that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. Interestingly, a detailed statistical diffusion analysis suggested two-component diffusion for cholesterol in the plasma membrane of live cells. One of these components was similar to a freely diffusing phospholipid analogue, whereas the other one was significantly faster. When a cholesterol analogue was localized to the outer leaflet only, the fast diffusion of cholesterol disappeared, and it diffused similarly to phospholipids. Overall, our results suggest that cholesterol diffusion in the cell membrane is heterogeneous and that this diffusional heterogeneity is due to cholesterol's nanoscale interactions and localization in the membrane.

show abstract

Section: Resultssupporting

confidence: 53%

Nanoscale dynamics of cholesterol in the cell membrane

Pinkwart

Schneider

Lukoseviciute

et al. 2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Zero-mode waveguides, nanopores and nanoapertures have received a large interest for many biophysics applications including molecular sensing, [55][56][57][58] DNA sequencing, 59-61 enzymatic reaction monitoring, 62,63 and biomembrane investigations. 64,65 With their ability to probe the protein tryptophan autofluorescence demonstrated here, new possibilities are offered to interrogate single proteins in their native state at physiological concentrations. Figure 1a presents the scheme of our experiment: a single ZMW milled in a 50 nm thick opaque aluminum film is positioned at the focus of a UV confocal microscope (the complete setup is detailed in the Supporting Information Fig.…”

mentioning

confidence: 99%

Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides

et al. 2019

Self Cite

View full text Add to dashboard Cite

Single molecule detection provides detailed information about molecular structures and functions, but it generally requires the presence of a fluorescent marker which can interfere with the activity of the target molecule or complicate the sample production. Detecting a single protein with its natural UV autofluorescence is an attractive approach to avoid all the issues related to fluorescence labelling.However, the UV autofluorescence signal from a single protein is generally extremely weak. Here, we use aluminum plasmonics to enhance the tryptophan autofluorescence emission of single proteins in the UV range. Zero-mode waveguides nanoapertures enable observing the UV fluorescence of single label-free β-galactosidase proteins with increased brightness, microsecond transit times and operation at micromolar concentrations. We demonstrate quantitative measurements of the local concentration, diffusion coefficient and hydrodynamic radius of the label-free protein over a broad range of zero-mode waveguide diameters. While the plasmonic fluorescence enhancement has generated a tremendous interest in the visible and near-infrared parts of the spectrum, this work pushes further the limits of plasmonic-enhanced single molecule detection into the UV range and constitutes a major step forward in our ability to interrogate single proteins in their native state at physiological concentrations.

show abstract

“…The transition regime predicted from the theoretical diffusion law for isolated domain organization was thus reported, and it allowed a refinement of the characteristic size of the nanometric membrane heterogeneities and a quantitative estimate of the surface area occupied by lipid-dependent nanodomains 39 . Alternatively, nanometric illumination has also been developed using near-field scanning optical microscopy 40 or planar optical nanoantennas 41 . More recently, combining stimulated emission depletion (STED) and FCS has provided a powerful and sensitive tool to document the diffusion law with very high spatial resolution.…”

Section: Representative Resultsmentioning

confidence: 99%